Horseshoe crab hemolymph contains an endotoxin-mediated serine protease pathway which causes coagulation of hemolymph in response to endotoxin (lipopolysaccharide) (reviewed in Iwanaga, Curr. Opin. Immunol. 5, 74-82, 1993). Limulus amebocyte lysate (LAL) is widely used in assays to detect endotoxin in a variety of test samples, including human and animal pharmaceuticals, biological products, and medical devices. Pharmaceuticals and research products are often tested for contamination from bacterial endotoxins. These contaminants can cause serious side effects if injected into the body, including fever and possibly death.
In an LAL assay, an extract of Limulus amebocytes is mixed with components which confer increased sensitivity and/or stability to the LAL, forming an LAL reagent. A test sample is mixed with the LAL reagent and compared with a series of known endotoxin standards mixed with LAL reagent. Sample and standard results are compared to determine the concentration of endotoxin in the sample.
The LAL assay for endotoxin is not entirely specific. Reaction also occurs in response to .beta.-1,3-glucans, which react with LAL through a separate glucan-sensitive enzymatic pathway (see Iwanaga, 1993). These .beta.-glucans are polymers of glucose, with varying molecular weights and, primarily, .beta.-1,3-glycosidic linkages between the glucose subunits. If a sufficient quantity of a .beta.-glucan is present in a test sample, a false-positive result occurs. A false-positive can result in a manufacturer unnecessarily discarding an expensive medical device or lot of product.
It can often be difficult to determine whether a positive result in the LAL test is due to endotoxin or to .beta.-glucan contamination. Thus, there is a continuing need in the art for endotoxin-specific assays which reduce or eliminate the incidence of false-positive results due to .beta.-glucan contamination.